Despite the undisputed fact that most of the vitamin D utilized by man is generated in the skin, little is known about either the tissue site of generation or the subcellular localization of the vitamin. Moreover, although the photometabolism of 7-dehydrocholesterol to previtamin D3 is now well understood, the role of other exogenous and endogenous factors, such as pigmentation and thermal factors, on both the generation and release of vitamin D is unknown. We intend to first localize vitamin D3 in various strata of the skin, and then within subcellular organelle fractions. The stratification experiments will be performed in both mouse and human skin, exploiting the unique intraepithelial cleaving capabilites of the staphylococcal exfoliation. Profiles of vitamin D precursors, present in the free sterol fractions of various tissue and subcellular fractions, will be analyzed by thin layer and high pressure liquid chromatography against authentic standards. The sites of vitamin D generation will be localized ultrastructurally by freeze-fracture cytochemistry soluble-tracer autoradiography, immunocytochemistry. Then, we intend to analyze the impact of varying certain exogenous and endogenous factors on both the quantities and localization of vitamin D in skin. We will analyze the role of pigmentation in albino vs. pigmented hairless mouse skin, the effect of incident ultraviolet light, influence of thermal factors, and removal of the stratum corneum reservoir on cutaneous Vitamin D generation. Further knowledge of the skin's role in vitamin D metabolism may lead to more rational methods for controlling cutaneous vitamin generation in clinical situations requiring its under- or over-production.